Biology subjects

Ruegg, M. A.

Publications and source records attributed to Ruegg, M. A..

12 recordsLinked to original sources

bioRxiv2026

Laminin-α2 is required for the maintenance of the myotendinous junction in vivo

The myotendinous junction (MTJ) is a critical interface between muscle fibers and tendons, essential for force transmission between muscle and bone. Laminin-2, a key extracellular matrix (ECM) component, is strongly enriched at this interface. Mutations in the LAMA2 gene cause LAMA2-related muscular dystrophy (LAMA2 MD), an early-onset severe congenital muscular dystrophy. Here, we examined the MTJ in dyW/dyWmice, a mouse model for LAMA2 MD. We find a strong disruption of MTJ morphology, including altered muscle fiber tips, collagen XXII mislocalization, and reduced muscle tendon interface. As MTJ loading is altered in dyW/dyW mice and MTJ maintenance requires loading and unloading, we also examined MTJ structures upon denervation-induced unloading. While muscle fiber tip morphology resembled that of dyW/dyW mice, collagen XXII distribution was not affected and the muscle-tendon interface was preserved. Finally, proteomic profiling via laser capture microdissection and mass spectrometry revealed significant regional and global shifts in MTJ protein composition in dyW/dyW and denervated mice. Across both models, we identified integrin-associated remodeling as a shared response linked to the perturbed muscle fiber tip morphology. These findings demonstrate that laminin-2 is required for MTJ stability, and that mechanical unloading contributes to the observed phenotype. Importantly, our results suggest that disruptions in MTJ structure and protein composition may contribute to the pathology observed in LAMA2 MD.

molecular biology↗

bioRxiv2026

c-MAF transduces motor neuron firing to sustain fast-glycolytic myofibers and neuromuscular junctions

This study examined how motoneuron activity influences transcription factor binding in mouse fast glycolytic Myh4+ muscle fibers. Single nucleus multiomics of innervated versus denervated tibialis anterior muscles revealed altered chromatin accessibility: SIX and c-MAF binding sites decreased while JUN, FOS, and RUNX1 sites increased in denervated Myh4+ myonuclei. c-MAF showed strong nuclear enrichment after 100 Hz stimulation and periods of increased motoneuron activity but was absent following denervation, establishing it as a primary readout of fast motoneuron firing. Genome-wide analysis demonstrated that c-MAF binding site spacing encodes functionally distinct muscle gene programs. Analysis of constitutive and inducible skeletal muscle-specific c-Maf mutants revealed that c-MAF loss caused region-specific MYH4+ fiber atrophy, MYH1/MYH2 fiber type shifts resembling ALS G93A mouse phenotypes, and progressive neuromuscular junction fragmentation with increased motoneuron terminal sprouting and ectopic reinnervation. These findings establish c-MAF as a critical mediator linking motoneuron activity to muscle gene regulation, fiber integrity, and neuromuscular junction maintenance in fast glycolytic fibers.

physiology↗

bioRxiv2025

Mini-agrin prevents calcium leakage and restores the dystrophin complex.

Muscle cell death in muscular dystrophies depends upon calcium ion (Ca++) leakage through sarcolemma and sarcoplasmic reticulum, triggered by muscle stretch during eccentric contraction. We show here that Ca++ spikes are detected in dystrophic myogenic cells in culture since early differentiation, before sarcomere assembly and contraction. Healthy and genetically corrected dystrophic myotubes do not display Ca++ spikes which are blocked by co-culturing DMD myogenic cells with embryonic mouse motoneurons or treating them with agrin proteoglycan. Same effect is elicited by a muscle spliced, COOH peptide of agrin (termed here mini-agrin) that interacts with dystroglycan, favouring its binding to the basal lamina. Lack of dystrophin in DMD myotubes results in decreased expression of CaV1.1 (CACNA1S), a Ca++ sensor component of the Dihydropyridine Receptor (DHPR) complex, known to regulate Ryanodine Receptor 1 (RyR1). These events explain the emergence of Ca++ spikes. Mini-agrin addition to medium, or lentivector-mediated mini-agrin expression in transplanted cells in vivo, stabilize the expression of CaV1.1 on the membrane. This leads to disappearance of Ca++ spikes and to reappearance of -dystroglycan, -sarcoglycan and n-NOS, indicating the reconstitution of the dystrophin complex in the absence of dystrophin. These findings unveil a novel regulatory mechanism and offer a new therapeutic opportunity for targeting calcium ion influx as a co-treatment strategy.

cell biology↗

bioRxiv2025

Dual AAV gene therapy using laminin-linking proteins ameliorates muscle and nerve defects in LAMA2-related muscular dystrophy

Adeno-associated virus (AAV)-mediated gene replacement holds promise for treating genetic diseases but faces challenges due to AAVs limited packaging capacity and potential immune responses to transgene products, especially in patients lacking endogenous protein. LAMA2-related muscular dystrophy (LAMA2 MD), a severe congenital disorder caused by loss of laminin-2, presents both hurdles: the LAMA2 gene exceeds AAV capacity, and severely affected patients do not produce the native protein. Here, we developed an AAV-based therapy using two engineered linker proteins derived from endogenously expressed components. These linker proteins restore laminin receptor binding and polymerization, enabling reassembly of a functional basement membrane. Dual AAV delivery of the linkers in a severe LAMA2 MD mouse model resulted in robust expression and significant improvements in muscle histology and function. Employing myotropic capsids enabled therapeutic efficacy at lower vector doses. However, muscle-specific targeting unmasked a LAMA2-related peripheral neuropathy. To address this, we expressed one linker under a muscle-specific promoter and the other under a ubiquitous promoter, delivered via AAV9 or AAV8. This approach achieved near-complete phenotypic restoration when administered neonatally and provided significant benefit when given at progressed disease stages. Our strategy offers a mutation-independent, size-compatible, and potentially immune-tolerable treatment for LAMA2 MD with broad clinical potential.

neuroscience↗

bioRxiv2024

Single-nuclei sequencing of skeletal muscle reveals subsynaptic-specific transcripts involved in neuromuscular junction maintenance

The neuromuscular junction (NMJ) is the synapse formed between motor neurons and skeletal muscle fibers. Its stability relies on the continued expression of genes in a subset of myonuclei, called NMJ myonuclei. Here, we use single-nuclei RNA-sequencing (snRNA-seq) to identify numerous undescribed NMJ-specific transcripts. To elucidate how the NMJ transcriptome is regulated, we also performed snRNA-seq on sciatic nerve transected, botulinum toxin injected and Musk knockout muscles. These data show that NMJ gene expression is not only driven by agrin-Lrp4/MuSK signaling, but is also affected by electrical activity and trophic factors other than agrin. By selecting three previously undescribed NMJ genes Etv4, Lrtm1 and Pdzrn4, we further characterize novel contributors to NMJ stability and function. AAV-mediated overexpression and AAV-CRISPR/Cas9-mediated knockout show that Etv4 is sufficient to upregulate expression of [~]50% of the NMJ genes in non-synaptic myonuclei, while muscle-specific knockout of Pdzrn4 induces NMJ fragmentation. Further investigation of Pdzrn4 revealed that it localizes to the Golgi apparatus and interacts with MuSK protein. Collectively, our data provide a rich resource of NMJ transcripts, highlight the importance of ETS transcription factors at the NMJ and suggest a novel pathway for NMJ post-translational modifications.

molecular biology↗

bioRxiv2024

Muscle fiber Myc is dispensable for muscle growth and forced expression severely perturbs homeostasis

The oncogenic transcription factor Myc stimulates many growth processes including cell cycle progression and ribosome biogenesis. Myc expression is low in adult skeletal muscle, but is upregulated upon growth stimuli. Furthermore, muscle fiber Myc overexpression recapitulates many aspects of growth-related gene expression, suggesting Myc may mediate pro-growth responses to anabolic stimuli, such as exercise. Here, we tested this hypothesis by examining mouse models in which Myc was specifically eliminated or overexpressed in skeletal muscle fibers or muscle stem cells (MuSC). While muscle fiber Myc expression increased during muscle growth and Myc expression in MuSCs was required for successful muscle regeneration, muscle fiber Myc expression was dispensable for post-natal, mechanical overload or PKB/Akt-induced muscle growth in mice. Similarly, constitutive Myc expression did not promote skeletal muscle hypertrophy, but instead impaired muscle fiber structure and function within days. These data question the role of Myc in skeletal muscle growth.

physiology↗

bioRxiv2023

Destabilization of neuromuscular junctions and deregulation of activity-dependent signalling pathways in Myotonic Dystrophy type I

Myotonic Dystrophy type I (DM1) is the most common muscular dystrophy in adults. Previous reports have highlighted that neuromuscular junctions (NMJs) deteriorate in skeletal muscle from DM1 patients and mouse models thereof. However, the underlying pathomechanisms and their contribution to muscle dysfunction remain unknown. We compared changes in NMJs and activity-dependent signalling pathways in HSALR and Mbnl1{Delta}E3/{Delta}E3 mice, two established mouse models for DM1. DM1 muscle showed major deregulation of calcium/calmodulin-dependent protein kinases II (CaMKIIs), which are key activity sensors regulating synaptic gene expression and acetylcholine receptor (AChR) recycling at the NMJ. Both mouse models displayed increased fragmentation of the endplate, which preceded muscle degeneration. Endplate fragmentation was not accompanied by changes in AChR turnover at the NMJ. However, expression of synaptic genes was up-regulated in DM1 muscle, which may be linked to the abnormally high activity of histone deacetylase 4 (HDAC4), a known target of CaMKII. Consistently, expression of myosin heavy chains was deregulated as well, leading to a major switch to type IIA fibres in Mbnl1{Delta}E3/{Delta}E3 muscle, and to a lesser extent in HSALR muscle. Interestingly, although HDAC4 was efficiently induced upon nerve injury, synaptic gene up-regulation was abrogated in DM1 muscle, together with a reduced increase in AChR turnover. This suggested that HDAC4-independent mechanisms lead to the defective response to denervation in DM1 muscle. Our study shows that activity-dependent signalling pathways are disturbed in DM1 muscle, which may contribute to NMJ destabilization and muscle dysfunction in DM1 patients.

physiology↗

bioRxiv2023

Fast, multiplexable and highly efficient somatic gene deletions in adult mouse skeletal muscle fibers using AAV-CRISPR/Cas9

Molecular screens comparing different disease states to identify candidate genes rely on the availability of fast, reliable and multiplexable systems to interrogate genes of interest. CRISPR/Cas9- based reverse genetics is a promising method to eventually achieve this. However, such methods are sorely lacking for multi-nucleated muscle fibers, since highly efficient nuclei editing is a requisite to robustly inactive candidate genes. Here, we couple Cre-mediated skeletal muscle fiber-specific Cas9 expression with myotropic adeno-associated virus-mediated sgRNA delivery to establish a system for highly effective somatic gene mutation in mice. Using well-characterized genes, we show that local or systemic inactivation of these genes copy the phenotype of traditional gene-knockout mouse models. Thus, this proof-of-principle study establishes a method to unravel the function of individual genes or entire signaling pathways in adult skeletal muscle fibers without the cumbersome requirement of generating knockout mice.

molecular biology↗

bioRxiv2022

Nerve pathology is prevented by linker proteins in mouse models for LAMA2-related muscular dystrophy

LAMA2-related muscular dystrophy (LAMA2 MD or MDC1A) is caused by mutations in the LAMA2 gene encoding laminin-2, the long chain of several heterotrimeric laminins. Laminins are essential components of the extracellular matrix that interface with underlying cells. The pathology of LAMA2 MD patients is dominated by the severe muscular dystrophy but also involves other tissues. In the dyW/dyW mouse model for LAMA2 MD, amelioration of muscle function by skeletal muscle-specific expression of the two linker proteins, mini-agrin and LNNd, is sufficient to greatly increase survival. In such survivors, the phenotype is dominated by the hindlimb paralysis. We now show that ubiquitous expression of the two linker proteins in dyW/dyW mice improves muscle function and prevents hindlimb paralysis. The same ameliorating effect of the linker proteins was seen in dy3K/dy3K mice, which represent the most severe mouse model of LAMA2 MD. In summary, these data show that the two linker proteins can compensate the loss of laminin-2 in many, if not all tissues affected in LAMA2 MD.

neuroscience↗

bioRxiv2021

Dual roles of mTORC1-dependent activation of the ubiquitin-proteasome system in muscle proteostasis

Muscle size is controlled by the PI3K-PKB/Akt-mTORC1-FoxO pathway, which integrates signals from growth factors, energy and amino acids to activate protein synthesis and inhibit protein breakdown. While mTORC1 activity is necessary for PKB/Akt-induced muscle hypertrophy, its constant activation alone induces muscle atrophy. Here we show that this paradox is based on mTORC1 activity promoting protein breakdown through the ubiquitin-proteasome system (UPS) by simultaneously inducing ubiquitin E3 ligase expression via feedback inhibition of PKB/Akt and proteasome biogenesis via Nuclear Factor Erythroid 2-Like 1 (Nrf1). Muscle growth was restored by reactivation of PKB/Akt, but not by Nrf1 knockdown, implicating ubiquitination as the limiting step. However, both PKB/Akt activation and proteasome depletion by Nrf1 knockdown led to an immediate disruption of proteome integrity with rapid accumulation of damaged material. These data highlight the physiological importance of mTORC1-mediated PKB/Akt inhibition and point to juxtaposed roles of the UPS in atrophy and proteome integrity.

physiology↗

bioRxiv2021

Distinct and additive effects of calorie restriction and rapamycin in aging skeletal muscle

As global life expectancy continues to climb, maintaining skeletal muscle function is increasingly essential to ensure a good life quality for aging populations. Calorie restriction (CR) is the most potent and reproducible intervention to extend health and lifespan, but is largely unachievable in humans. Therefore, identification of "CR mimetics" has received much attention. CR targets nutrient-sensing pathways centering on mTORC1. The mTORC1 inhibitor, rapamycin, has been proposed as a potential CR mimetic and is proven to counteract age-related muscle loss. Therefore, we tested whether rapamycin acts via similar mechanisms as CR to slow muscle aging. Contrary to our expectation, long-term CR and rapamycin-treated geriatric mice display distinct skeletal muscle gene expression profiles despite both conferring benefits to aging skeletal muscle. Furthermore, CR improved muscle integrity in a mouse with nutrient-insensitive sustained muscle mTORC1 activity and rapamycin provided additive benefits to CR in aging mouse muscles. Therefore, RM and CR exert distinct, compounding effects in aging skeletal muscle, opening the possibility of parallel interventions to counteract muscle aging.

physiology↗

bioRxiv2019

mTORC1 signaling is not essential for the maintenance of muscle mass and function in adult sedentary mice

BackgroundThe balance between protein synthesis and degradation (proteostasis) is a determining factor for muscle size and function. Signaling via the mammalian target of rapamycin complex 1 (mTORC1) regulates proteostasis in skeletal muscle by affecting protein synthesis and autophagosomal protein degradation. Indeed, genetic inactivation of mTORC1 in developing and growing muscle causes atrophy resulting in a lethal myopathy. However, systemic dampening of mTORC1 signaling by its allosteric inhibitor rapamycin is beneficial at the organismal level and increases lifespan. Whether the beneficial effect of rapamycin comes at the expense of muscle mass and function is yet to be established.\n\nMethodsWe conditionally ablated the gene coding for the mTORC1-essential component raptor in muscle fibers of adult mice (iRAmKO). We performed detailed phenotypic and biochemical analyses of iRAmKO mice and compared them with RAmKO mice, which lack raptor in developing muscle fibers. We also used polysome profiling and proteomics to assess protein translation and associated signaling in skeletal muscle of iRAmKO mice.\n\nResultsAnalysis at different time points reveal that, as in RAmKO mice, the proportion of oxidative fibers decreases, but slow-type fibers increase in iRAmKO mice. Nevertheless, no significant decrease in body and muscle mass, or muscle fiber area was detected up to 5 months post-raptor depletion. Similarly, ex vivo muscle force was not significantly reduced in iRAmKO mice. Despite stable muscle size and function, inducible raptor depletion significantly reduced the expression of key components of the translation machinery and overall translation rates.\n\nConclusionsRaptor depletion and hence complete inhibition of mTORC1 signaling in fully-grown muscle leads to metabolic and morphological changes without inducing muscle atrophy even after 5 months. Together, our data indicate that maintenance of muscle size does not require mTORC1 signaling, suggesting that rapamycin treatment is unlikely to negatively affect muscle mass and function.

molecular biology↗